Accumulating evidence suggests that microbiota plays an important role in modulating lifespan. This makes possible to use symbiotic bacteria as “living drugs”, which live in the host organism and promote its longevity. We propose to create bacteria, which dramatically extend lifespan of its host. Such bacteria have to produce not one, but a set of longevity-promoting substances with optimal concentrations and dynamics of secretion. To obtain such bacteria we propose to use directed evolution, a process that mimics Darwinian selection on a laboratory scale. This approach has never been applied to drug development before. Directed evolution enables simultaneous modulation of a number of bacterial metabolic pathways andsubsequent selection of the most effective longevity-promoting variants. Experiments will be conducted on a model system consisting of C.elegans and its intestinal symbiont E.coli. Due to highly conserved aging pathways, obtained bacteria may be further used to develop longevity-promoting human drug.
Bacteria E.coli serve as the food source for C.elegans, but at particular stages of the nematode life course they can also exist as intestinal symbionts. Moreover, it was shown that E.coli influence physiology and lifespan of C.elegans. It was revealed that several mutations in E.coli genome increase or vice versa reduce the nematode lifespan.
The relationship between C.elegans and E.coli at different stages of the life course of the nematode
Left picture – During development, bacteria mainly serve as a source of food for C.elegans
Center picture – In adult worms some bacteria are not digested and become symbionts
Right picture – As the worm ages, bacteria proliferating within the lumen of the gut become detrimental to the host (Cabriero and Gems, 2013)
The project aims to create E.coli strains that are able to extend the lifespan of C.elegans. For this purpose we propose to employ directed evolution – a process that mimics Darwinian selection on a laboratory scale. At first, phenotypic diversity of E.coli is generated using global transcription machinery engineering (gTME) approach. The gTME randomly alters key proteins regulating the global transcriptome and generates a new type of diversity at the transcriptional level. Then a set of bacterial strains with reprogrammed transcriptome (a bacterial library) is created and E.coli strains which demonstrate the highest ability to extend C.elegans lifespan are selected. We propose to perform not less than 2 cycles of gTME.
For gTME we propose to alter genes of transcription initiation factors σ, which regulate expression of hundreds of genes. At the first cycle of gTME the gene which encodes the main sigma factor σ70 (RpoD) is subjected to random mutagenesis. At the second cycle of gTME the gene of sigma factor σ38 (RpoS), which regulates expression of stationary phase genes, is targeted for mutation.
The selected bacteria are studied to identify transcriptome, proteome and metabolome modifications which result in longevity-promoting phenotype.
Creation of symbiotic bacteria, which are able to increase the lifespan of the host, and study of biological mechanisms underlying these longevity-promoting interactions.
I stage – Creation of bacterial strains which extend the lifespan of C.elegans
(duration – 1,5 years)
1) Construction of library of E.coli strains with reprogrammed transcriptome
- Random mutagenesis of σ-factor gene using error-prone PCR.
- Cloning of obtained sequences into plasmids and transformation into E.coli.
2) Selection of E.coli strains which demonstrate the highest ability to extend C.elegans lifespan
- C.elegans are raised on one of the bacterial mutant strains and analyzed for their lifetime. 1500-2000 strains from mutant library are screened.
- Selection of E.coli strains which demonstrate the highest ability to extend C.elegans lifespan.
2nd cycle of gTME using the gene of another σ-factor – iteration of experiments 1)-2)
II Stage – Study of E.coli strains which extend the lifespan of C.elegans
(duration – 1 year)
- Analysis of transcriptome, proteome and metabolome of E.coli strains which promote longevity of C.elegans.
- Identification of E.coli genes and biological pathways which affect the lifespan of C.elegans.
- Development of longevity-promoting “living drug” based on symbiotic bacteria.
- Demonstration of the possibility of directed evolution to create symbiotic bacteria with such a complex phenotype as ability to extend the host lifespan.
- Identification of new genes and biological pathways of coli which affect C.elegans longevity.